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The microsomal fraction isolated from dog mesenteric nerve fibres was found to contain ATPase activity stimulated by micromolar concentrations of Ca ions. Such a high-affinity Ca-ATPase (hereafter referred to as HA Ca-ATPase) followed a Michaelis-Menten kinetics with K for Ca ions of 0.4 μM and V=12.5±2.4 μmol P.mgh. The examination of the subcellular origin of HA Ca-ATPase revealed that this enzyme is associated with axonal plasma membranes as documented by its co-purification with several plasma membrane marker enzymes and with tetrodotoxin-sensitiveH-saxitoxin binding. The addition of exogenous magnesium ions (Mg) resulted in a non-competitive inhibition of HA Ca-ATPase with K=0.5 mM. The reaction velocity of HA Ca-ATPase was also inhibited by other divalent ions with the order of potency Mg>Mn >Zn≥Co>Ni. In contrast to low affinity (high K) Mg- and Ca-ATPase, the HA Ca-ATPase was insensitive to the inhibition by sodium azide (10 mM) and sodium fluoride (10 mM). Similarly, the specific activity of HA Ca-ATPase was unaffected by vanadate (100 μM) and N-ethylmaleinimide (100 μM). It is concluded that axonal plasma membranes of dog mesenteric nerves contain HA Ca-ATPase which seems to be unrelated to calcium-transporting Mg-dependent, Ca-stimulated ATPase. [ABSTRACT FROM AUTHOR]

A high affinity Ca/Mg ATPase has been identified and localized in synaptic membrane subfractions. This enzyme is stimulated by low concentrations of Ca (≤1 μM) believed to approximate the range of Ca in the synaptosomal cytosol (0.1 to 5.0 μM). The opiate agonist levorphanol, in a concentration-dependent fashion, inhibited Ca-stimulated ATP hydrolysis in lysed synaptic membranes. This inhibition was reversed by naloxone, while dextrorphan, the inactive opiate isomer, was without effect. Inhibition by levorphanol was most pronounced in a subfraction of synaptic membranes (SPM-1). The inhibition of Ca-stimulated ATP hydrolysis was characterized by a reduction in V for Ca. Levorphanol pretreatment reduced the Hill coefficient (H) of 1.5 to 0.7, suggesting cooperative interaction between the opiate receptor and the enzyme protein. Levorphanol, but not dextrorphan, also inhibited (28%) ATP-dependent Ca uptake by synaptic membranes. Opiate ligand stereoisomers were tested for their effects on calmodulin stimulating of high affinity Ca/Mg ATPase in synaptic membranes. Levorphanol (10 μM), but not the inactive stereoisomer (+)dextrorphan, significantly inhibited (35%) the calmodulin-activated Ca-dependent ATP hydrolysis activity in a preparation of lysed synaptic membranes. Both Ca-dependent and calmodulin-dependent stimulation of the enzyme in the presence of optimal concentrations of the other co-substrate were inhibited by levorphanol (35-40%) but not dextrorphan. Inhibition of ATP hydrolysis was characterized by a reduction in V for both Ca and calmodulin stimulation of the enzyme. Calmodulin stimulation of enzyme activity was most pronounced in SPM-1, the membrane fraction which also exhibits the maximal opiate inhibition (40%) of the Ca-ATPase. The results demonstrate that opiate receptor activation inhibits a high affinity Ca/Mg ATPase in synaptic plasma membranes in a stereospecific fashion. The inhibition of the enzyme may occur by a mechanism involving both Ca and calmodulin. Inhibition of calmodulin activation may contribute to the mechanism by which opiate ligands disrupt synaptosomal Ca buffering mechanisms. Changes in the cytosolic distribution of synaptosomal Ca following inhibition of Ca/Mg ATPase may underlie some of the pharmacological effects of opiate drugs. [ABSTRACT FROM AUTHOR]

Procedures have been developed which allow the preparation of highly pure endoplasmic reticulum and plasma membrane from tendrils of Bryonia dioica. These and further membrane fractions were used to study vanadate-sensitive ATPase activity as well as MgATP-driven transport of Ca. Calcium-translocating ATPases were detected in the endoplasmic reticulum, the plasma membrane and the mitochondrial fraction and characterized kinetically and with respect to the effects of various inhibitors. The endoplasmic-reticulum Ca-translocating ATPase was stimulated by KCl and was calmodulin-dependent. The plasma-membrane enzyme was not affected by these agents. These, as well as the inhibitor data, show that the Ca-translocating ATPases of the endoplasmic reticulum and the plasma membrane are distinctly different enzymes. Upon mechanical stimulation, the activities of the vanadate-sensitive K, Mg-ATPase and the Ca-translocating ATPase(s) increased rapidly and transiently, indicating that increasing transmembrane proton and calcium fluxes are involved in the early stages of tendril coiling. [ABSTRACT FROM AUTHOR]

Intracellular Ca levels in Paramecium must be tightly controlled, yet little is understood about the mechanisms of control. We describe here indirect evidence that a phosphoenzyme intermediate is the calmodulin-regulated plasma membrane Ca pump and that a Ca-ATPase activity in pellicles (the complex of cell body surface membranes) is the enzyme correlate of the plasma membrane pump protein. A change in Ca pump activity has been implicated in the chemoresponse of paramecia to some attractant stimuli. Indirect support for this is demonstrated using mutants with different modifications of calmodulin to correlate defects in chemoresponse with altered Ca homeostasis and pump activity. [ABSTRACT FROM AUTHOR]

The activity of Ca-ATPase in the inner ear of the guinea pig was studied ultracytochemically by the lead citrate reaction. The electron-dense reaction products as an expression of Ca-ATPase activity were localized in endolymphatic cells of Reissner's membrane, in outer and inner hair cells and in some supporting cells. The main finding was the difference in the localization of Ca-ATPase in outer and inner hair cells. In the latter cells the activity sites were mainly intracellular and in apical membrane specializations, whereas in the outer hair cells the enzyme was localized in the apical membrane specializations and the basolateral plasma membrane. [ABSTRACT FROM AUTHOR]

A sensitive enzyme-linked immunoadsorbant assay was developed to quantify Ca -ATPase and calsequestrin from sarcoplasmic reticulum in human muscle biopsies. Tissue levels of Ca -ATPase and calsequestrin averaged 51.5 ± 28.1 and 6.4 ± 1.8mg/g muscle protein, respectively, in control muscles (means ± SD, n=12). The high sensitivity and specificity of the antibodies make the assay a useful tool in the diagnosis of human neuromuscular disorders where defects in sarcoplasmic reticulum function may be expected. The assay was applied to muscle biopsies from patients with myotonia congenita and paramyotonia congenita Eulenburg. The calsequestrin concentration was normal in all patient muscles. The Ca -ATPase content was also within the normal range but varied considerably with the percentage distribution of slowtwitch fibres. This indicates that the prolonged relaxation observed in the muscles of patients with these disorders is not caused by faulty expression of Ca-ATPase and calsequestrin. [ABSTRACT FROM AUTHOR]

Enzyme-cytochemical studies were performed to clarify the pathogenesis of myocardial damage in endotoxemia. The reaction product of Ca-ATPase was localized on the pinocytotic vesicles and plasma membrane of endothelial cells of capillaries and sarcolemma, sarcoplasmic reticulum and mitochondria of cardiomyocytes in control rats. However, the enzyme reaction product was reduced in amount and irregularly distributed on the plasma membrane, and on the increased number of pinocytotic vesicles of the endothelial cells of capillaries and the sarcolemma and sarcoplasmic reticulum of edematous cardiomyocytes after endotoxin injection. It is concluded that endotoxin induces structural changes and decreased activity in Ca-ATPase in the vascular endothelium and myocytes of the heart. [ABSTRACT FROM AUTHOR]

Assays for complete quantification of Na, K-and Ca-ATPase in crude homogenates of rat ventricular myocardium by determination of K-and Ca-dependent p-nitrophenyl phosphatase ( pNPPase) activities were evaluated and optimized. Using these assays the total K-and Ca-dependent pNPPase activities in ventricular myocardium of 11-12 week-old rats were found to be 2.98±0.10 and 0.29±0.02 μmol×min×g wet wt. (mean±SEM) ( n=5), respectively. Coefficient of variance of interindividual determinations was 7 and 12%, respectively. The total Na, K-and Ca-ATPase concentrations were estimated to 2 and 10 nmol×g wet wt., respectively. Evaluation of a putative developmental variation revealed a biphasic age-related change in the rat myocardial Ca-dependent pNPPase activity with an increase from birth to around the third week of life followed by a decrease. By contrast, the K-dependent pNPPase activity of the rat myocardium showed a decrease from birth to adulthood. It was excluded that the changes were simple out-come of variations in water and protein content of myocardium. Expressed per heart, the K-and Ca-dependent pNPPase activity gradually increased to a plateau. The present assay for Na, K-ATPase quantification has the advantage over [H] ouabain binding of being applicable on the ouabain-resistant rat myocardium, and is more simple and rapid than measurements of K-dependent 3- 0-methylfluorescein phosphatase (3- 0-MFPase) in crude tissue homogenates. Furthermore, with few modifications the pNPPase assay allows quantification of Ca-ATPase on crude myocardial homogenates. Age-dependent changes in K-and Ca-dependent pNPPase activities are of developmental interest and indicate the importance of close age match in studies of quantitative aspects of Na, K-and Ca-ATPase in excitable tissues. [ABSTRACT FROM AUTHOR]

In an attempt to clarify the mechanism of sarcoplasmic reticulum (SR) dysfunction during the genesis of irreversible damage in the ischemic-reperfused myocardium, the changes in SR Ca-pumping ATPase (Ca-activated, Mg-dependent ATPase; Ca-ATPase) activity were studied during ischemia and subsequent reperfusion in the isolated perfused guinea pig heart preparation and correlated with the accumulation of calcium in the myocardium. Although the SR Ca-ATPase activity was not affected by ischemia of 40 min, reperfusion of the ischemic myocardium resulted in a definite time-dependent decrease in the enzyme activity. The reduction of SR Ca-ATPase activity was associated with a concomitant decrease in the enzyme concentration in the isolated SR and was in a good correlation with a substantial accumulation within the mhocardium. As the results indicated the possibility that proteolytic degradation by a calcium-activated protease(s) was responsible for the reduction of enzyme activity, we examined for the possible involvement of calcium-activated neutral protease (CANP). However, SR Ca-ATPase obtained either from the normal hearts or from the hearts after 40-min ischemia was found to be quite resistant to proteolytic actions of the two forms of CANP, i.e., μCANP and mCANP partially purified from the guinea pig heart. These results suggest that a destructive process leading to the degradation of SR Ca-ATPase is activated by reperfusion, but not by ischemia per se, and that CANP is not implicated in the degradation of SR Ca-ATPase. [ABSTRACT FROM AUTHOR]

Effects of oxygen free radicals on Ca/Mg ATPase and ATP-independent Ca-binding activities were examined in rat heart sarcolemma. Membranes were incubated with different oxygen radical generating media such as xanthine + xanthine oxidase, hydrogen peroxide, and hydrogen peroxide + Fe. In the presence of xanthine + xanthine oxidase, Ca ATPase activity was stimulated and this effect was prevented by the addition of superoxide dismutase. Hydrogen peroxide also showed a significant increase in Ca-ATPase activity in a dose-dependent manner and this effect was blocked by catalase. On the other hand, a combination of hydrogen peroxide + Fe decreased Ca-ATPase activity; this depression was prevented by the addition of D-mannitol. The observed change in Ca-ATPase activity due to oxygen free radicals was associated with changes in V, whereas Ka remained unaffected. Both xanthine + xanthine oxidase and hydrogen peroxide increased whereas, hydrogen peroxide + Fe inhibited the ATP-independent Ca-binding activities. It is suggested that oxygen free radicals may influence Ca movements in the cell by altering the Ca/Mg ATPase and Ca-binding activities of the membrane and these effects may be oxygen-radical species specific. [ABSTRACT FROM AUTHOR]

Calcium is released from the isolated heavy sarcoplasmic reticulum (SR) of frog skeletal muscle upon application of 0.1-1 mM diethylpyrocarbonate (DEP, an imidazolyl reagent). The Ca-ATPase activity of SR was suppressed by 20% in the presence of 1 mM DEP. More than 1 mM of free magnesium ion or 5 μM ruthenium red eliminated the effect of DEP on calcium release but not on Ca-ATPase activity. A plausible site of DEP action is on the calcium channel. [ABSTRACT FROM AUTHOR]

Thapsigargin (TG) and cyclopiazonic acid (CPA) have been reported to be potent inhibitors of the sarcoplasmic reticulum (SR) Ca uptake in isolated SR vesicles and cells. We have examined the effect of TG and CPA on (1) the Ca uptake by the SR in saponinskinned rat ventricular trabeculae, using the amplitude of the caffeine-induced contraction to estimate the Ca content loaded into the SR, (2) the spontaneous Ca oscillations at pCa 6.6 using force oscillation as the indicator, and (3) the myofilament Ca sensitivity in Triton X-100-treated preparations. Inhibition of Ca loading by TG and CPA increased with time of exposure to the inhibitor over 18-24 min. TG and CPA produced half inhibition of Ca loading at 34.9 and 35.7 μM respectively, when 18-24 min were allowed for diffusion. The spontaneous force oscillations were more sensitive to the inhibitors: 10 μM TG and 30 μM CPA both abolished the oscillations in this time. The myofilament Ca sensitivity was not affected by 10 and 300 μM TG or CPA. The results show that the concentrations of TG and CPA necessary to inhibit the SR Ca uptake of skinned ventricular trabeculae are much higher than the reported values for single intact myocytes. One reason for this may be slow diffusion of the inhibitors into the multicellular trabecula preparation. [ABSTRACT FROM AUTHOR]

Circular smooth muscle strips isolated from cat gastric fundus were studied in order to understand whether the sarcoplasmic reticulum (SR) and SR Ca-ATPase could play a role in the regulation of the muscle tone. Cyclopiazonic acid (CPA), a specific inhibitor of SR Ca-ATPase, caused a significant and sustained increase in muscle tone, depending on the presence of extracellular Ca. Nifedipine and cinnarizin only partially suppressed the CPA-induced tonic contraction. Bay K 8644 antagonized the relaxant effect of nifedipine in CPA-contracted fundus. Nitric-oxide-releasing agents sodium nitroprusside and 3-morpholino-syd-nonimine completely suppressed the CPA-induced tonic contraction. The blockers of Ca-activated K channels, tetraethylammonium, charybdotoxin and/or apamin, decreased the contractile effect of CPA. Vanadate increased the tone but did not change significantly the effect of CPA. CPA exerted its contractile effect even when Ca influx was triggered through the Na/Ca exchanger and the other Ca entry pathways were blocked. Thapsigargin, another specific SR Ca-ATPase inhibitor, also increased the muscle tone. The effect of thapsigargin was completely suppressed by sodium nitroprusside and 3-morpholino-sydnonimine and partially by nifedipine. In conclusion, under conditions when the SR CaATPase is inhibited, the tissue develops a strong tonic contraction and a large part of this is mediated by Ca influx presumably via nifedipine-sensitive Ca channels. This study suggests the important role of SR Ca-ATPase in the modulation of the muscle tone and the function of SR as a 'buffer barrier' to Caentry in the cat gastric fundus smooth muscle. [ABSTRACT FROM AUTHOR]

Ca-ATPase activity was studied ultra-cytochemically in the middle ear mucosa of the guinea pig. On electron microscopic examination, the most intense reaction was found on the microvilli. Reaction products were also observed on the cilia and around and between the secretory granules on the apical side of the cells in their secretory phase. The basolateral membranes contained few reaction products, while very little or no activity was found on the basal membrane. [ABSTRACT FROM AUTHOR]

Ca-ATPase activity was examined cytochemically in the lateral cochlear wall of the guinea pig. The reaction products showing Ca-ATPase activity were found along the folded plasma membrane of the strial marginal cells. In contrast, little or no reaction was seen on the apical surfaces of these cells. There were also marked reaction products on the microvilli and the endolymphatic cell surface of Reissner's membrane, and the apical and lateral plasma membranes of the spiral prominence and the external sulcus cells. These reactions completely disappeared when Ca or ATP was removed from the incubation medium. Our results strongly suggest that Ca-ATPase plays an important role in Ca transport system for the regulation of Ca concentration in the cochlear endolymph. [ABSTRACT FROM AUTHOR]

We used ultracytochemistry to examine Ca-ATPase activity in the vestibular epithelia of the guinea pig. Many reaction products were found along the basolateral plasma membrane of the vestibular dark cell. There were also marked reaction deposits on the apical and lateral cell membranes of the transitional cells, and the utricular and saccular wall cells. Both sensory and supporting cells showed Ca-ATPase activity along their ciliary membrane and apical-lateral cell surfaces. Our findings indicate that the Ca-ATPase activity found on the plasma membrane is closely related to Ca-transport across the plasma membrane. When either Ca or ATP was omitted from the incubation medium, enzyme activity (as seen by the staining reaction present) was completely abolished. Our present results suggest that Ca-ATPase located in the vestibular epithelia plays a significant role in the regulation of the Ca-concentration in the vestibular endolymph. [ABSTRACT FROM AUTHOR]

The in vitro effect of porcine insulin on Na+K-, Ca- and Mg-ATPases of the rat erythrocyte membrane of normal and alloxan-induced diabetic rats was investigated. Na+K- and Ca-stimulated enzyme activities were significantly decreased in diabetic rats in comparison to normal animals. The specific activities of both these ATPases in the latter group were markedly reduced on pre-incubating the ghosts with insulin. Similar treatment of the erythrocyte membranes of diabetic animals, however, resulted in a significant increase of these activities. These qualitatively different effects of the hormone in the two groups increased progressively with hormone concentration and duration of pre-incubation. Mg-stimulated ATPase activity was not significantly affected in diabetes or by insulin. [ABSTRACT FROM AUTHOR]

Observations that suggest a metabolic basis for the association between asthma and rhinitis and their pathogenesis are reviewed. We have observed increased plasma lysophosphatidylcholine (LPC) levels and decreased (Na-K)-ATPase activity in the leukocytes of patients with asthma and rhinitis. In asthmatic patients leukocytes also showed significantly lower Ca-ATPase activity. LPC increases cell membrane permeability to Na and Ca, causes membrane depolarization, potentiates IgE response, promotes phagocytic activity, inhibits adenylate cyclase, and stimulates phosphodiesterase, thus decreasing cAMP levels in the cells. These changes, when accompanied by failure of Na/K and Ca pumps, would lead to increased cytosolic Ca and enhanced release of mediators from the mast cells in the airway lumen, producing a state of airway inflammation. We have also observed abnormal fall in specific airway conductance at residual volume in these patients, thereby showing small airway obstruction in asthma as well as in rhinitis. This could be due to airway inflammation caused by mechanisms described above. Large airway obstruction seen in asthma could be due to failure of Ca pump, normal activity of which seems essential for proper efflux of Ca from airway smooth muscle cells. We suspect that increased plasma LPC levels and failure of the homeostatic ionic pumps occur due to some defect in oxidative metabolism and suggest that asthma and rhinitis could be seen as metabolic disorders. [ABSTRACT FROM AUTHOR]

The Ca2(+)-ATPase from rat liver microsomes has been solubilized in Triton X-100 and purified to homogeneity by ficoll-sucrose treatment, column chromatography with agarose-hexane adenosine 5'-triphosphate Type 2, and high pressure liquid chromatography (HPLC). The purified enzyme obtained by this sequential procedure exhibited a 183-fold increase in specific activity. After ficoll-sucrose treatment, the activity of the Ca2(+)-ATPase was stable for at least two weeks when stored at -70 degrees C. In SDS-polyacrylamide gels, several fractions from HPLC chromatography showed a single band at a position corresponding to a molecular weight of about 107 kDa. This value is consistent with the molecular weight of the phosphoenzyme intermediate of endoplasmic reticulum (ER) Ca2(+)-ATPase. Further characterization of the ER Ca2(+)-ATPase was performed by western immunoblots. Antiserum raised against the 100-kDa sarcoplasmic reticulum (SR) Ca2(+)-ATPase cross-reacted with the purified Ca2(+)-ATPase from rat liver ER membranes. [ABSTRACT FROM AUTHOR]

Calpain I purified from human erythrocyte cytosol activates both the ATP hydrolytic activity and the ATP-dependent Ca2+ transport function of the Ca2(+)-translocating ATPase solubilized and purified from the plasma membrane of human erythrocytes and reconstituted into phosphatidylcholine vesicles. Following partial proteolysis of the enzyme by calpain I, both the initial rates of calcium ion uptake and ATP hydrolysis were increased to near maximal levels similar to those obtained upon addition of calmodulin. The proteolytic activation resulted in the loss of further stimulation of the rates of Ca2+ translocation or ATP hydrolysis by calmodulin as well as an increase of the affinity of the enzyme for calcium ion. However, the mechanistic Ca2+/ATP stoichiometric ratio was not affected by the proteolytic treatment of the reconstituted Ca2(+)-translocating ATPase. The proteolytic activation of the ATP hydrolytic activity of the reconstituted enzyme could be largely prevented by calmodulin. Different patterns of proteolysis were obtained in the absence or in the presence of calmodulin during calpain treatment: the 136-kDa enzyme was transformed mainly into a 124-kDa active ATPase fragment in the absence of calmodulin, whereas a 127-kDa active ATPase fragment was formed in the presence of calmodulin. This study shows that calpain I irreversibly activates the Ca2+ translocation function of the Ca2(+)-ATPase in reconstituted proteoliposomes by producing a calmodulin-independent active enzyme fragment, while calmodulin antagonizes this activating effect by protecting the calmodulin-binding domain against proteolytic cleavage by calpain. [ABSTRACT FROM AUTHOR]

The influence of the asymmetric addition of various divalent cations and protons on the properties of active Ca transport have been examined in intact human red blood cells. Active Ca efflux was determined from the initial rate ofCa loss after CoCl was added to block Ca loading via the ionophore A23187. Ca-ATPase activity was measured as phosphate production over 5 min in cells equilibrated with EGTA-buffered free Ca in the presence of A23187. The apparent Ca affinity of active Ca efflux ( K=30−40 μmol/liter cells) was significantly lower than that measured by the Ca-ATPase assay ( K=0.4 μ m). Possible reasons for this apparent difference are considered. Both active Ca efflux and Ca-ATPase activity were reduced to less than 5% of maximal levels (20 mmol/liter cells · hr) in Mg-depleted cells, and completely restored by reintroduction of intracellular Mg. Active Ca efflux was inhibited almost completely by raising external CaCl (but not MgCl) to 20 mm, probably by interaction of Ca at the externally oriented EP conformation of the pump. Cd was more potent than Ca in this inhibition, while Mn was less potent and 10 mm Ba was without effect. A Ca: proton exchange mechanism for active Ca efflux was supported by the results, as external protons (pH 6-6.5) stimulated active Ca efflux at least twofold above the efflux rate at pH 7.8 Ca transport was not affected by decreasing the membrane potential across the red cell. [ABSTRACT FROM AUTHOR]

Human erythrocyte Ca2+-translocating ATPase was solubilized from calmodulin-depleted membranes using the detergent Triton X-100, and subsequently purified by calmodulin-affinity chromatography. The purified enzyme was reconstituted in artificial phospholipid vesicles using a cholate-dialysis method and various phospholipids. The reconstituted enzyme was able to translocate Ca2+ inside the vesicles, both in the absence and in the presence of the Ca2+-chelating agent, oxalate, inside the vesicles. The tightness of coupling between ATP hydrolysis and cation translocation was investigated by the use of different ionophoretic compounds. The efficiency of Ca2+ translocation was measured by the ability of the ionophores to stimulate ATP hydrolytic activity of the reconstituted enzyme. It was found that the maximum stimulation of the ATP hydrolytic activity was induced by the electroneutral Ca2+/2H+ ionophore A23187 (9 to 10-fold). A Ca2+ ionophore unable to translocate H+, CYCLEX-2E, was less efficient in stimulating the activity of the reconstituted enzyme (two- to threefold). However, the combined addition of CYCLEX-2E plus protonophores further increased the ATP hydrolytic activity (around fourfold), whereas, the protonophores did not further stimulate ATP hydrolysis in the presence of A23187. Furthermore, in the absence of Ca2+ ionophore, the electroneutral K+(Na+)/H+ ionophoretic exchanger, monensin, stimulated the rate of ATP hydrolysis in the reconstituted enzyme two- or threefold, respectively. These results suggest that the Ca2+-ATPase not only translocates Ca2+ but also H+ in the opposite direction. [ABSTRACT FROM AUTHOR]

Basolateral plasma membranes from rat kidney cortex have been purified 40-fold by a combination of differential centrifugation, centrifugation in a discontinuous sucrose gradient followed by centrifugation in 8% percoll. The ratio of leaky membrane vesicles (L) versus right-side-out (RO) and inside-out (IO) resealed vesicles appeared to be L∶RO∶IO=4∶3∶1. High-affinity Ca-ATPase, ATP-dependent Ca transport and Na/Ca exchange have been studied with special emphasis on the relative transport capacities of the two Ca transport systems. The kinetic parameters of Ca-ATPase activity in digitonin-treated membranes are: K=0.11 μ m Ca and V=81±4 nmol P/min·mg protein at 37°C. ATP-dependent Ca transport amounts to 4.3±0.2 and 7.4±0.3 nmol Ca/min·mg protein at 25 and 37°C, respectively, with an affinity for Ca of 0.13 and 0.07 μ m at 25 and 37°C. After correction for the percentage of IO-resealed vesicles involved in ATP-dependent Ca transport, a stoichiometry of 0.7 mol Ca transported per mol ATP is found for the Ca-ATPase. In the presence of 75 mm Na in the incubation medium ATP-dependent Ca uptake is inhibited 22%. When Na is present at 5 mm an extra Ca accumulation is observed which amounts to 15% of the ATP-dependent Ca transport rate. This extra Ca accumulation induced by low Na is fully inhibited by preincubation of the vesicles with 1 mm ouabain, which indicates that (Na−K)-ATPase generates a Na gradient favorable for Ca accumulation via the Na/Ca exchanger. In the absence of ATP, a Na gradient-dependent Ca uptake is measured which rate amounts to 5% of the ATP-dependent Ca transport capacity. The Na gradient-dependent Ca uptake is abolished by the ionophore monensin but not influenced by the presence of valinomycin. The affinity of the Na/Ca exchange system for Ca is between 0.1 and 0.2 μ m Ca, in the presence as well as in the absence of ATP. This affinity is surprisingly close to the affinity measured for the ATP-dependent Ca pump. Based on these observations it is concluded that in isolated basolateral membranes from rat kidney cortex the Ca-ATPase system exceeds the capacity of the Na/Ca exchanger four- to fivefold and it is therefore unlikely that the latter system plays a primary role in the Ca homeostasis of rat kidney cortex cells. [ABSTRACT FROM AUTHOR]

The ATP-dependent accumulation of Ca2+ by sarcoplasmic reticulum vesicles at 37 degrees reaches a peak after approximately 100 sec. The Ca2+-loading level then declines until a steady-state level is reached which is 20% less than the peak value. This spontaneous release of Ca2+ is enhanced by inclusion of maleate in the Ca2+ uptake medium. Increasing the extravesicular osmolarity by the addition of sucrose to the Ca2+ uptake medium prevents spontaneous Ca2+ release and increases the steady-state Ca2+-loading capacity of sarcoplasmic reticulum vesicles. Swelling of sarcoplasmic reticulum vesicles during Ca2+ uptake in medium containing sucrose is indicated by changes in the light-scattering intensity. These experiments indicate that the capacity of sarcoplasmic reticulum vesicles to accumulate Ca2+ is limited by the osmotic gradient generated by the increase in intravesicular Ca2+. Swelling of sarcoplasmic reticulum vesicles during Ca2+ uptake causes spontaneous Ca2+ release. [ABSTRACT FROM AUTHOR]

The regulation of the guinea-pig pancreatic acinar plasma membrane Ca pump by protein kinase A, protein kinase C and calmodulin was investigated. The results were compared with the effects of these regulators on the high affinity Ca-ATPase found in this membrane preparation. The catalytic subunit of cyclic AMP-dependent protein kinase stimulated Ca transport 2-fold, but had no effect on Ca-dependent ATPase activity. Purified protein kinase C, the phorbol ester 12-O-tetradecanoyl phorbol-13-acetate and diacylglycerol derivative, 1-stearoyl-2-arachidonoyl- sn-glycerol, failed to stimulate the Ca-uptake but augmented the Ca-dependent ATPase activity. Exogenously added calmodulin failed to stimulate either activity. In addition, two antagonists of calmodulin activity, trifluoperazine and compound 48/80 produced a concentration-dependent inhibition of Ca-transport. These data suggest the presence of endogenous calmodulin within guinea-pig pancreatic acinar plasma membranes. Both calmodulin antagonists failed to influence the Ca-dependent ATPase activity. The ability of boiled extracts from guinea-pig pancreatic acinar plasma membranes to stimulate the Ca-ATPase activity in calmodulin-depleted erythrocyte plasma membranes confirmed the presence of endogenous calmodulin. Our results imply a role for calmodulin and cAMP-dependent protein kinase, but not protein kinase C, in the regulation of Ca efflux from pancreatic acinar cells. These results also provide further evidence suggesting that the high affinity Ca-ATPase does not catalyze the plasma membrane Ca-transport activity observed in pancreatic acini. [ABSTRACT FROM AUTHOR]

Effects of endotoxin administration on the ATP-dependent Ca transport in canine cardiac sarcolemma were investigated. The results show that the sidedness of the sarcolemmal vesicles was not affected but the ATP-dependent Ca transport in cardiac sarcolemma was decreased by 22 to 46% (p < 0.05) at 4 h following endotoxin administration. The kinetic analysis indicates that the V for ATP and for Ca were decreased by 50% (p < 0.01) and 32% (p < 0.01), respectively, while the Km values for ATP and Ca were not significantly affected after endotoxin administration. Magnesium (1-5 mM) stimulated while vanadate (0.25-3.0 μM) inhibited the ATP-dependent Ca transport, but the Mg-stimulated and the vanadate-inhibitable activities remained significantly lower in the endotoxin-treated animals. These data demonstrate that endotoxin administration impairs the ATP-dependent Ca transport in canine cardiac sarcolemma and that the impairment is associated with a mechanism not affecting the affinity towards ATP and Ca. Additional experiments show that the Ca sensitivity of the Ca-ATPase activity was indifferent between the control and endotoxic groups suggesting that endotoxic injury impairs Ca pumping without affecting Ca-ATPase activity. Since sarcolemmal ATP-dependent Ca transport plays an important role in the regulation of cytosolic Ca homeostasis, an impairment in the sarcolemmal ATP-dependent Ca transport induced by endotoxin administration may have a pathophysiological significance in contributing to the development of myocardial dysfunction in endotoxin shock. [ABSTRACT FROM AUTHOR]

Partially purified plasma membrane fractions were prepared from guinea-pig pancreatic acini. These membrane preparations were found to contain an ATP-dependent Ca-transporter as well as a heterogenous ATP-hydrolytic activity. The Ca-transporter showed high affinity for Ca (K = 0.04 ± 0.01 μM), an apparent requirement for Mg and high substrate specificity. The major component of ATPase activity could be stimulated by either Ca or Mg but showed a low affinity for these cations. At low concentrations, Mg appeared to inhibit the Ca-dependent ATPase activity expressed by these membranes. However, in the presence of high Mg concentration (0.5-1 mM), a high affinity Ca-dependent ATPase activity was observed (K = 0.08 ± 0.02 μM). The hydrolytic activity showed little specificity towards ATP. Neither the Ca-transport nor high affinity Ca-ATPase activity were stimulated by calmodulin. The results demonstrate, in addition to a low affinity Ca (or Mg)-ATPase activity, the presence of both a high affinity Ca-pump and high affinity Ca-dependent ATPase. However, the high affinity Ca-ATPase activity does not appear to be the biochemical expression of the Ca-pump. [ABSTRACT FROM AUTHOR]

The Ca uptake activity of rat cardiac sacroplasmic reticulum (CSR) in ventricular homogenates is highly unstable, and this instability probably accounts for the low specific activity of Ca uptake in previously reported fractions of isolated rat CSR. The instability was observed at either 0° or 37°, but the Ca uptake activity was relatively stable at 25°. The decay of Ca uptake activity at 0° could not be prevented by either PMSF or leupeptin, but dithiothreitol exerted some protective effects. Sodium metabisulfite prevented decay of the Ca uptake activity of homogenates kept on ice but not of homogenates kept at 37°. We also found that release of the CSR from the cellular debris required homogenization in high KCI. This distinguishes rat CSR from canine CSR. Isolated CSR was produced by a combination of differential centrifugation and discontinuous sucrous gradient centrifugation. The average rate of the sustained oxalate-supported calcium uptake in the resulting CSR fraction was 0.36 μmol/min-mg in the absence of CSR calcium channel blockers and 0.67 μmol/min/mg in the presence of 10 μM ruthenium red. Thus, this preparation has the advantage of containing both the releasing and non-releasing fractions of the CSR. The Ca-ATPase rates averaged 1.07 μmol/min/mg and 0.88 μmol/min-mg in the absence and presence of ruthenium red, respectively. Although these rates are higher than previously reported rates, this CSR preparation should still be considered a 'crude' preparation. A major distinction between the rat CSR and dog CSR was the lower content of Ca-ATPase in rat CSR, as judged by SDS-PAGE. Preparations of CSR isolated by this method may be useful in evaluating alterations in CSR function. [ABSTRACT FROM AUTHOR]

The goat spermatozoa membranes isolated after treatment with octa (ethylene glycol) mono n-dodecyl ether (CE) followed by discontinuous sucrose density gradient centrifugation have been found to contain an ATPase that is stimulated by externally added Ca only. The membrane fraction has also found to contain Mg-dependent Ca-ATPase activity, however the former activity is about 2 fold higher than the latter. The molecular weight of the enzyme is found to be about 97,000 on SDS-polyacrylamide gel. The optimum concentration of Ca required for maximum activity is 3 mM for both Mg-dependent and Mg-independent Ca-ATPase. Histidine and imidazole buffers are found to be the most suitable for dependent and independent enzyme activities respectively. ATP with an optimum concentration of 4 mM is observed to be the best substrate than any other nucleotides. The inhibitors like trifluoperazine and vanadate and group specific probes e.g. DTNB and TNBS inhibit these two enzymes but at different rates. Ca-uptake study shows that the uptake in the presence of Ca and ATP is higher than in the presence of Mg, Ca and ATP. The findings lead us to believe that the Mg-independent Ca-ATPase has some role in Ca transport like Mg-dependent enzyme. [ABSTRACT FROM AUTHOR]

We have compared properties of the red blood cell Ca-ATPase in two types of preparations: red cell membrane ghosts (enzyme in unfractionated membranes) and after purification (detergent-soluble enzyme). The Ca-ATPase activity was studied with respect to its requirement for: calmodulin, calcium, magnesium, monovalent cations, ionic strength, pH, and temperature. Sensitivity of the Ca-ATPase activity in the two preparations to anticalmodulin drugs and to engineered calmodulins with amino acid substitutions was determined. Finally, stoichiometry of the formation of phosphorylated enzyme intermediate (EP) and titrations of the ATP binding region with fluorescein 5′-isothiocyanate (FITC) were characterized. For the first time a high phosphorylation level of 2.0-2.4 mmol EP/mg of purified enzyme is reported. The two enzyme preparations have been found to be very similar with respect to the dependency of all the regulating factors described here. These results complement findings reported from various laboratories on the similarity of other kinetic properties as well as the similarity of modulation of the Ca-ATPase activity by phospholipids and proteolysis in the membranous and purified enzyme. Thus, the purified detergent-soluble enzyme is very well suited for kinetic characterization of the red cell Ca-ATPase. [ABSTRACT FROM AUTHOR]

A method is described for monitoring intracellular ionized calcium (Ca) and determining kinetic and thermodynamic parameters of Ca-extrusion from intact lymphocytes. The method uses ratiometric spectrofluorometry and the fluorescent Ca dye indo-1. Lymphocytes were loaded with calcium and placed in a low calcium medium. A novel formula for calculation of intracellular Ca that corrects for background fluorescence and fluorescence quenching was used. Calcium extrusion resulted in exponential decrease in cytoplasmic Ca with a rate constant of 0.031 ± 0.003 sec, maximal rate of 23 ± 7 nM/sec, dissociation constant of 366 ± 63 nM, Hill coefficient of 2.3 ± 0.4, Q of 2.58 ± 0.28, and activation energy of 18.3 Kcal/mol. This method should allow for characterization of the Ca-extrusion system of lymphocytes and may be applicable to other blood cell types. [ABSTRACT FROM AUTHOR]

Preparations of cardiac sarcoplasmic reticulum (CSR) isolated from the rat by differential centrifugation have been widely used for measuring alterations in intracellular calcium flux in response to metabolic and pharmacologic disruptions. However, the purity of these SR fractions has not been firmly established. Using a combination of differential and linear sucrose gradient centrifugation, we have isolated rat CSR with high specific activity and purity. By SDS-PAGE analysis, the preparation is enriched in a protein (110 kD) of similar size to the Ca-ATPase of SR from other sources. Gels stained with the dye 'Stains All' reveal a blue colored 55 kD band, confirming the presence of calsequestrin, the intraluminal low-affinity calcium binding protein of SR. The presence of the transmembrane 53 kD glycoprotein of SR was confirmed by endoglycosidase-H treatment followed by SDS-PAGE and also by a modified Western blotting technique. The rate of calcium uptake in this preparation averages 130 nmol/mg over the first minute of accumulation, approximately 4 times that previously reported for rat CSR. Calcium uptake in our preparation was essentially complete within 5 minutes. Preparations isolated by this method should be of value in future studies measuring alterations in rat CSR function. [ABSTRACT FROM AUTHOR]

The Ca binding site region of the Ca - ATPase of skeletal muscle sarcoplasmic reticulum was labeled with several fluorescent analogs of dicyclohexylcarbodiimide. As has been shown by Chadwick and Thomas [1, 2], in the absence of Ca in the medium, labeling with the naphthyl carbodiimide results in the inhibition of enzyme activity. Further, Ca occupancy of the high affinity sites of the enzyme protects against incorporation into the site(s). The fluorescent carbodiimide has been used to determine the depth of the site of label incorporation relative to the aqueous-bilayer interfaces by quenching studies using spin-labeled fatty acid derivatives. The series of quenchers used have their spin-label moiety located at different positions along the fatty acid chain. It was found that after suitable correction for differences in partitioning of the various derivatives, the order of quenching efficiency was 16 - > 12- > 10- > 7- > 5-NS, indicating that the naphthyl moiety is near the center of the bilayer. In contrast, quenching with the aqueous-restricted I indicated that the label is accessible from the external milieu, likewise for a presumed aqueous quencher, acrylamide. The aqueous quenchers accessibilities were altered upon Ca binding to the ATPase. Quenching of the intrinsic fluorescence with the x-NS derivatives indicates that the ATPase tryptophan residues are primarily localized at the aqueous-membrane interfaces, with the order of quenching being 5- > 7- > 10- > 12- > 16-NS. The trp residue(s) which changes its fluorescence upon Ca binding is shown to be near the membrane surface. [ABSTRACT FROM AUTHOR]

The two high affinity calcium binding sites of the cardiac (Ca + Mg)-ATPase have been identified with the use of Eu. Eu competes for the two high affinity calcium sites on the enzyme. With the use of laser-pulsed fluorescent spectroscopy, the environment of the two sites appear to be heterogeneous and contain different numbers of HO molecules coordinated to the ion. The ion appears to be occluded even further in the presence of ATP. Using non-radiative energy transfer studies, we were able to estimate the distance between the two Ca sites to be between 9.4 to 10.2 A in the presence of ATP. Finally, from the assumption that the calcium site must contain four carboxylic side chains to provide the 6-8 ligands needed to coordinate calcium, and based on our recently published data, we predict the peptidic backbone of the two sites. [ABSTRACT FROM AUTHOR]

The mechanisms of allosteric regulation of the Ca-ATPases of cardiac and skeletal sarcoplasmic reticulum by ATP have been compared. Although both enzymes showed stimulation of ATPase activity by ATP, the cardiac enzyme did not show the plateau in ATPase activity at 10-100μM ATP seen with the skeletal enzyme. Likewise the phosphoenzyme (EP) levels did not plateau with the cardiac enzyme as they did with the skeletal enzyme. The apparent negative cooperatively which was seen in the kinetics of ATP hydrolysis at low ATP concentrations was not due to negative cooperatively in substrate binding to either enzyme. The cardiac enzyme did show, however, much higher affinity for the ATP analog, AMPPCP, which helps explain how AMPPCP blocks ATPase activity in the cardiac enzyme and stimulates ATPase activity in the skeletal enzyme. Fluorescein isothiocyanate was used to determine if allosteric regulation takes place through site-site interactions in oligomers. The 1 to 1 ratio between AMPPCP binding sites and FITC binding sites eliminated allosteric regulation by effector sites in both enzymes. The allosteric mechanism which remained was one in which the active-site becomes an effector-site by the early departure of ADP in the reaction mechanism. The step stimulated by the binding of ATP at the active-site turned effector-site was a nonphosphorylated form of the enzyme in cardiac sarcoplasmic reticulum and a phosphorylated form in skeletal sarcoplasmic reticulum. [ABSTRACT FROM AUTHOR]

A critical review of the experimental literature concerning the metabolism of all-cis-4, 7, 10, 13, 16, 19-docosahexaenoate-containing phospholipids in muscle and retina suggests that it plays an essential role in maximizing the Ca/ATP stoichiometry of the Ca-ATPase of sarcoplasmic reticulum and retinal photoreceptor disks. Docosahexaenoate-phosphatidylcholine is proposed to participate in oligomerization of Ca-ATPase necessary for the establishment of a high Ca/ATP coupling ratio of the Ca pump in these tissues. Possible tests of this hypothesis are presented. [ABSTRACT FROM AUTHOR]

The polycationic dyes, Hoechst 33342 (Bisbenzimide,2′-(4-ethoxyphenyl)-5-(4-methyl-1-piperazinyl) 2,5′-bi 1H benzimidazole) and Hoechst 33258 (Bisbenzimide,2′-(4-hydroxyphenyl) 5-(4-methyl-1-piperazinyl)-2,5′-bi-1H-benzimidazole) alter the activity of the sarcoplasmic reticulum Ca channel. Although they act competitively, Hoechst 33342 decreases, while Hoechst 33258 increases, the rate of channel-mediated Ca efflux from junctional sarcoplasmic reticulum vesicles. Unlike other cationic sarcoplasmic reticulum Ca channel antagonists, Hoechst 33342 blocks the ryanodine-activated Ca channel. Both Hoechst 33342 and Hoechst 33258 inhibit the channel incorporated into the planar lipid bilayer. Since the only structural difference between the two dyes is that the agonist Hoechst 33258 has a hydroxy group where the antagonist Hoechst 33342 has an ethoxy group, it is possible that the more hydrophobic, bulky ethoxy group blocks Ca movement through the channel, whereas the hydroxy group only reduces the rate of Ca movement. The opinions or assertions contained herein are private ones of the author ad are not to beconstrued as official or reflecting the views of the Department of Defense or the Uniformed Services University of the Health Sciences. [ABSTRACT FROM AUTHOR]

The inhibition of Ca-ATPase, (Na+K)-ATPase and Na/Ca exchange by Cd was studied in fish intestinal basolateral plasma membrane preparations. ATP driven Ca uptake into inside-out membrane vesicles displayed a K for Ca of 88±17 nm, and was extremely sensitive to Cd with an IC of 8.2±3.0 pM Cd, indicating an inhibition via the Ca site. (Na+K)-ATPase activity was half-maximally inhibited by micromolar amounts of Cd, displaying an IC of 2.6±0.6 μ m Cd. Cd ions apparently compete for the Mg site of the (Na +K)-ATPase. The Na/Ca exchanger was inhibited by Cd with an IC of 73±11 n m. Cd is a competitive inhibitor of the exchanger via an interaction with the Ca site (K = 11 n m). Bepridil, a Na site specific inhibitor of Na/Ca exchange, induced an additional inhibition, but did not change the K of Cd. Also, Cd is exchanged against Ca, albeit to a lesser extent than Ca. The exchanger is only partly blocked by the binding of Cd. In vivo cadmium that has entered the enterocyte may be shuttled across the basolateral plasma membrane by the Na/Ca exchanger. We conclude that intracellular Cd ions will inhibit plasma membrane proteins predominantly via a specific interaction with divalent metal ion sites. [ABSTRACT FROM AUTHOR]

Organotin compounds have been shown to interfere with cardiovascular system. We have studied the in vitro and in vivo effects of tributyltin bromide (TBT), triethyltin bromide (TET) and trimethyltin chloride (TMT) on the cardiac SR Ca pump, as well as on protein phosphorylation of SR proteins, in order to understand the relative potency of these tin compounds. All the three tin compounds inhibited cardiac SRCa uptake and Ca-ATPase in vitro in a concentration-dependent manner. The order of potency for Ca-ATPase as determined by IC, is TBT (2 μM) > TET (63 μM) > TMT (280 μM). ForCa uptake, it followed the same order i.e., TBT (0.35 μM) > TET (10 μM) > TMT (440 μM). In agreement with the in vitro results, both SR Ca-ATPase andCa uptake were significantly inhibited in rats treated with these tin compounds, indicating that these tin compounds inhibit cardiac SR Ca transport. cAMP significantly elevated (70-80%) theP-binding to SR proteins in vitro in the absence of any organotin. In the presence of organotins, cAMP-stimulatedP-binding to proteins was significantly reduced, but the decrease was concentration dependent only at lower concentrations. The order of potency is TBT > TET > TMT. In agreement with in vitro studies, cAMP-dependentP bound to proteins was significantly reduced in rats treated with TBT, TET and TMT. SDS-polyacrylamide gel electrophoresis of the cardiac SR revealed at least 30 Coomassie blue stainable bands ranging from 9 to 120 kDa. Autoradiographs from samples incubated in the presence of cAMP indicatedP incorporation in seven bands. Of these, the band corresponding to about 24 kDa molecular weight protein decreased in its intensity with the treatment of organotins. These results suggest that triorganotins may be affecting Ca pumping mechanisms through the alteration of phosphorylation of specific proteins in rat cardiac SR. [ABSTRACT FROM AUTHOR]

Ca-ATPase activity was solubilized, partly purified, and separated from nonspecific alkaline phosphatase activity (APase) of dentinogenically active rat incisor odontoblasts. Attempts were made to extract the enzymes by various agents, such as Triton X-100, deoxycholate, butanol, EDTA, and buffers of decreasing ionic strength. Solubilization by butanol followed by extraction with low concentrations of EDTA proved to be most effective. Purification and separation were done by molecular sieve chromatography. Ca-ATPase showed no activity against p-nitrophenyl phosphate ( p-NPP) or inorganic pyrophosphate (PP) and was unaffected by R 8231 [(±)-6(m-bromophenyl)-5,6-dihydroimidazo(2,1-b)thiazole oxalate]. It was activated by Ca and Mg ions in equimolar concentrations with the substrate. The enzyme was rapidly inactivated in the solubilized state. An apparent molecular weight of about 18,000 was obtained from molecular sieve data. APase, showing activity against ATP, PP, and p-NPP, was virtually totally inhibited by R 8231. It was activated by Mg ions but slightly reduced in activity by Ca ions. It had an apparent mol. wt. of 79,000. The results provide direct evidence for earlier suggestions of the existence in hard tissue forming cells of two phosphatases active at alkaline pH. [ABSTRACT FROM AUTHOR]

Two-week-old white leghorn chicks were fed a diet containing BAPN (0.05%) for three weeks. Thirty-six hours before sacrifice, the controls and BAPN fed chicks were dosed with S. The zone of provisional calcification was isolated, and S incorporation was estimated by liquid scintillation counting. Alkaline phosphatase and Ca-ATPase were biochemically analyzed. Microdensitometry, to assess the level of mineralization, was done on epiphysis and the metaphysis. Morphometry was performed on the various zones of growth plate. S incorporation was significantly lower in the bones of BAPN treated chicks as compared to the controls. The enzymatic studies showed a significant inhibition of alkaline phosphatase and Ca-ATPase. The microdensitometric studies showed a smaller area of highly mineralized bone in the zones of provisional calcification of the BAPN treated chicks as compared to the controls. Morphometry showed a reduction in the width of the zone of calcification in BAPN treated chicks as compared to the controls. On the basis of the above data, it is suggested that BAPN induced inhibition of mineralization during endochondral ossification may be the result of a lower synthesis of sulfur containing GAG's, the inhibition of enzymes alkaline phosphatase and Ca-ATPase and the derangement of cellular zones of the growth plate. The implications of these results lie in the fact than mineralization is dependent on multifactorial control of the microenvironment of bone and cartilage. [ABSTRACT FROM AUTHOR]

Two groups of weanling Sprague-Dawley rats were fed a low-selenium basal diet (Se 0.009 mg/kg) and the same diet supplemented with sodium selenite (Se 0.25 mg/kg), respectively, for 1, 2, and 3 months. At each feeding time, the Ca-ATPase activity, Ca uptake rate and the capacity of Ca uptake in isolated cardiac sacroplasmic reticulum from the Se-deficient rats were decreased significantly compared to those from the Se-supplemented rats, the contents of lipid peroxide in postmitochondrial supernatant and isolated sarcoplasmic reticulum from the Se-deficient rats were significantly higher than that from Se-supplemented rats. Compared to the Se-supplemented rats, the cytosolic glutathione peroxidase activity in Se-deficient rats decreased significantly. In addition, significant linear negative correlations of lipid peroxide in postmitochondrial supernatant to sarcoplasmic reticular Ca-ATPase activity, Ca uptake rate and to whole blood selenium concentration were observed. The results suggest that the enhancement of lipid peroxidation via the depressed glutathione peroxidase activity might be responsible for the decrease of Ca-ATPase and Ca uptake activities in sarcoplasmic reticulum in Se-deficient animals. [ABSTRACT FROM AUTHOR]

The distribution of actin filaments in pyloric gland cells of cattle was studied with respect to their functional significance in the process of exocrine secretion by use of rhodamine-phalloidin labelling and immunogold-electron microscopy based on the biotinstreptavidin bridge technique. Actin concentrates on the filamentous network of the luminal cell cortex. Membranes of secretory vesicles accumulating in the cell cortex are also labelled for actin. The present results support the concept of a barrier function of cortical microfilaments entrapping vesicles and linking them to the cytoskeleton. In addition, intracellular localization of calcium-ATPase activity was determined. Enzyme activity associated with the microfilamentous cortical matrix is supposed to be of cytoskeletal nature indicating participation of myosin (-like) structures in the dynamic secretion event. Deposition on the interior aspect of secretory vesicle membranes points to an ATPase transporting calcium into these organelles and enabling them to participate via storage of the cation in intracellular calcium homeostasis, thereby influencing the functional architecture of the cortical cytoskeleton. [ABSTRACT FROM AUTHOR]

HeLa cells in a monolayer culture were synchronized to S, G and mitotic phases by use of excess (2.5 mM) deoxythymidine double-block technique. The localizations of Ca-activated adenosine triphosphatase (ATPase) at different phases of the cell cycle were studied using light and electron-microscopic histochemical techniques, and microphotometric comparisons of the densities of reaction products. Enzyme reaction product was always localized in the endoplasmic reticulum, nuclear membrane, mitochondria and Golgi apparatus, but there were qualitative and quantitative differences related to the phases of the cell cycle. In S phase the activity was mainly concentrated in a perinuclear area of the cytoplasm whereas in G and mitosis the activity was scattered throughout the cell. The total activity per cell was maximal in G, was less in S phase and least in mitosis. Activity in the mitochondria and endoplasmic reticulum was distinctly less in mitosis than in other phases of the cell cycle. The mitochondrial ATPase differed from the ATPase at other sites in ion dependence and sensitivity to oligomycin. The results suggest that there may be several distinct ATPases in proliferating cells. [ABSTRACT FROM AUTHOR]

The effects of the calmodulin blocker, trifluoperazine (TEP), on membrane-bound Ca -ATPase, Na -K -ATPase (EC 3.6.1.3.) and the ultrastructure of the enamel organ were investigated in the lower incisors of normal and TFP-injected rats. The rats, of about 100 g body weight, were given either 0.2 ml physiological saline or 100 μg TFP dissolved in 0.2 ml physiological saline through a jugular vein and fixed by transcardiac perfusion with a formaldehyde-glutaraldehyde mixture at 1 and 2 h after TFP administration. Non-decalcified sections of the enamel organ less than 50 μm in thickness, prepared from dissected lower incisors, were processed for the ultracytochemical demonstration of Ca-ATPase and Na-K -ATPase by the one-step lead method at alkaline pH. In control saline-injected animals the most intense enzymatic reaction of Ca-ATPase was demonstrated along the plasma membranes of the entire cell surfaces of secretory ameloblasts. Moderate enzymatic reaction was also observed in the plasma membranes of the cells of stratum intermedium and papillary layer. Reaction precipitates of Na-K-ATPase activity were localized clearly along the plasma membranes of only the cells of stratum intermedium and papillary layer. The most drastic effect of TFP was a marked disappearance of enzymatic reaction of Ca-ATPase from the plasma membranes of secretory ameloblasts, except for a weak persistent reaction in the basolateral cell surfaces of the infranuclear region facing the stratum intermedium. The cells of stratum intermedium and papillary layer, however, continued to react for Ca-ATPase even after TFP treatment. Similarly, Na-K-ATPase activity in these cells was not inhibited by TFP administration. Ultrastructural examination of secretory ameloblasts revealed that administration of TFP caused no considerable cytological changes and did not act as a cytotoxic agent. These results suggest that secretory ameloblasts may have an active Ca transport system, which is modulated by an endogenous calmodulin. [ABSTRACT FROM AUTHOR]

Ca-ATPase activity (cf. Ando et al. 1981) was examined both light- and electron-microscopically in the neurohypophysis of the guinea pig. Apart from a strong activity within the walls of the blood vessels, in the parenchyma of the neurohypophysis the reaction product of the Ca-ATPase activity was restricted to the plasmalemma of the pituicytes. This reaction was completely dependent upon Ca and the substrate, ATP; the reaction was inhibited by 0.1 mM quercetin, an inhibitor of Ca-ATPase. A reduction of the enzyme activity occurred by 1) adding Mg to the standard incubation medium, and 2) substituting Ca with Mg at varing concentrations. In all experiments the neurosecretory fibers were devoid of Ca-ATPase activity. The function of the Ca-ATPase activity in the plasmalemma of the pituicytes is discussed in connection with the regulation of the extracellular Ca concentration, which seems to be important with respect to the discharge of secretory material from the neurosecretory fibers. [ABSTRACT FROM AUTHOR]

Ca-ATPase activity was demonstrated histochemically at light- and electron-microscopic levels in inner and outer segments of retinal photoreceptor cells of the guinea pig with the use of a newly developed one-step lead-citrate method (Ando et al. 1981). The localization of ouabain-sensitive, K-dependent p-nitrophenylphosphatase (K-NPPase) activity, which represents the second dephosphorylative step of the Na-K-ATPase system, was studied by use of the one-step method newly adapted for ultracytochemistry (Mayahara et al. 1980). In retinal photoreceptor cells fixed for 15 min in 2% paraformaldehyde the electron-dense Ca-ATPase reaction product accumulated significantly on the inner membranes of the mitochondria but not on the plasmalemma or other cytoplasmic elements of the inner segments. The membranes of the outer segments remained unstained except the membrane arrays in close apposition to the retinal pigment epithelium. The cytochemical reaction was Ca- and substrate-dependent and showed sensitivity to oligomycin. When Mg-ions were used instead of Ca-ions, a distinct reaction was also found on mitochondrial inner membranes. In contrast to the localization of the Ca -ATPase activity, the K-NPPase activity was demonstrated only on the plasmalemma of the inner segments, but not on the mitochondria, other cytoplasmic elements or the outer segment membranes. This reaction was almost completely abolished by ouabain or by elimination of K from the incubation medium. [ABSTRACT FROM AUTHOR]

Ca-ATPase activity was studied ultracytochemically (cf. Ando et al. 1981) in the paraphysis cerebri of the frog. An intense reaction was demonstrated on the plasmalemma of the microvilli at the apical pole of paraphyseal cells; in contrast, the basolateral plasmalemma showed only a slight staining. In addition, mitochondria, gap junctions, cilia, and cytoplasmic elements (e.g., microfilaments) displayed Ca-ATPase activity. Variation of the Ca-concentration in the incubation medium from 0.1 mM to 100 mM altered the Ca-ATPase activity of the cell organelles. The substitution of Ca-by Mg-ions resulted in a conspicuous decrease in the enzyme activity, especially on the apical plasmalemma. Ca-ATPase activity is claimed to be involved in a number of extra-and intracellular functions. In comparison to the epithelium of the adjacent choroid plexus the paraphyseal epithelial cell is thought to be a principal Ca-ion regulator of the cerebrospinal fluid in frogs. [ABSTRACT FROM AUTHOR]